Uses of thaxtomin and thaxtomin compositions as herbicides

ABSTRACT

There is a need for a selective, low-risk herbicide that can be used to control weeds in cereal cultures and turf. The present invention discloses that a bacterial secondary metabolite, thaxtomin and optionally another herbicide is an effective herbicide on broadleaved, sedge and grass weeds. Thaxtomin A and structurally similar compounds can be used as natural herbicides to control the germination and growth of weeds in cereal, turf grass, Timothy grass and pasture grass cultures with no phytotoxicity to these crops. As a natural, non-toxic compound, thaxtomin can be used as a safe alternative for weed control in both conventional and organic farming and gardening systems.

PRIORITY CLAIM

This application claims priority from U.S. application Ser. No.61/142,179, filed Dec. 31, 2008 and 61/261,504 filed Nov. 16, 2009 under35 USC 119(e), in which the contents of both are incorporated herein byreference. The application also claims priority from Taiwan Patentapplication no. 098144895, filed Dec. 25, 2009 under 35 USC 119(a)-(d),the contents of which are also incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions and methods for controlling thegermination and growth of broadleaf, sedge and grass weeds usingcompounds comprising thaxtomin, a cyclic dipeptide produced byStreptomyces sp., as an active ingredient.

BACKGROUND OF THE INVENTION

Natural products are substances produced by microbes, plants, and otherorganisms. Microbial natural products offer an abundant source ofchemical diversity, and there is a long history of utilizing naturalproducts for pharmaceutical purposes. However, secondary metabolitesproduced by microbes can also be successfully used for weed and pestcontrol in agricultural applications.

Thaxtomins (4-nitroindol-3-yl-containing 2,5-dioxopiperazines) are afamily of dipeptide phytotoxins produced by plant-pathogenicStreptomyces sp. (S. scabies, S. acidiscabies) that cause scab diseasesin potato (Solanum tuberosum) (King, Lawrence et al. 1992). Toxinproduction occurs in diseased tissue and can also be elicited in vitroin an optimal growth medium containing oat bran (Loria, Bukhalid et al.1995; Beauséjour, Goyer et al. 1999). King and her coworkers (King,Lawrence et al. 2001) demonstrated that all plant pathogenic species inthe Streptomyces family produce one or more thaxtomins with herbicidalactivity. Hiltunen et al. (Hiltunen, Laakso et al. 2006) purified fourthaxtomin analogs (thaxtomin A, thaxtomin A ortho isomer, thaxtomin Band thaxtomin D) from cultures of S. scabies and S. turbidiscabies andshowed that all four compounds induced similar symptoms of reduced shootand root growth, root swelling, (at 10-200 ppb) and necrosis (at200-1000 ppb) on micropropagated in vitro cultures of potato. Inaddition, thaxtomins applied in combinations, showed additive effects,but no synergism (Hiltunen, Laakso et al. 2006). According to Duke etal. (Duke, Baerson et al. 2003), both thaxtomin A (FIG. 1) and thaxtominD have marked activity as pre and post emergent, non-systemicherbicides, and concentrations of less than 1 uM of thaxtomin A causescell swelling, necrosis and growth inhibition in mono and dicotyledonousseedlings (Healy, Wach et al. 2000). Thaxtomin has been evaluated as anherbicide by Dow Agro Sciences, Inc., and while active, it lackedsystemic action (King, Lawrence et al. 2001). The presence of the nitrogroup in the indole ring required for an L,L-configuration of thediketopiperazine appears to be the minimal requirement forphytotoxicity. The position of the nitro group in the indole ring isvery site specific, and the phenyl portion of the phenylalanine plays anecessary role in structural requirements of phytotoxicity (King,Lawrence et al. 1989; King, Lawrence et al. 1992; King, Lawrence et al.2003). The herbicidal mode of action is based on disruption of cell wallsynthesis (Fry and Loria 2002), with inhibition of cellulosebiosynthesis being the main target (King et al., 2001; Duval et al.,2005; Johnson et al. 2007). Recently, Kang et al. (Kang, Semones et al.2008) have described the use of thaxtomin and thaxtomin compositions asalgaecides to control algae in water environments.

SUMMARY OF THE INVENTION

The present invention discloses the use of thaxtomin as a pre orpost-emergence herbicide against most common weeds in the cereal,pasture grass, Timothy grasses and turf grass growth systems. A “growthsystem” may be any ecosystem for growing cereal, pasture grass, Timothygrass and turf grass. For example, a “cereal growth system” may be acereal growth culture or may be a field containing planted cereal cropsor cereal seeds. Similarly, a “turf grass growth system” may be a turfgrass growth culture or may be a field, lawn or golf course containingplanted turf grass or turf grass seeds. It can serve as a saferalternative to synthetic herbicides now on the market. A primary objectof the invention is to provide novel herbicidal compositions againstboth broadleaf, sedge and grassy weeds, which include but are notlimited to Chenopodium album, Abutilon theophrasti, Helianthus annuus,Ambrosia artemesifolia, Amaranthus retroflexus, Convolvulus arvensis,Brassica kaber, Taraxacum officinale, Solanum nigrum, Malva neglect,Setaria lutescens, Bromus tectorum, Poa annua, Poa pratensis, Loliumperenne L. var. Pace, Festuca arundinaceae Schreb. var. Aztec II, AnthemII, LS1100, Echinochloa crus-galli, and particularly,Lambsquarter—Chenopodium album, Redroot Pigweed—Amaranthus retroflexus,Wild Mustard—Brassica kaber, Dandelion—Taraxacum officinale, and BlackNightshade—Solanum nigrum, that contains thaxtomin as an activeingredient. Another object is to provide a safe, non-toxic herbicidalcomposition that does not harm cereal crops, pasture grass, Timothygrass or turf grass and a method that will not harm the environment.

The above and other objects are accomplished by the present inventionwhich is directed to herbicidal compositions containing at least oneherbicidal agent, e.g., thaxtomin with optionally certain carriers tocontrol the growth and germination of weeds in the cereal growth systemand/or turf grass growth system and/or Timothy grass growth systemand/or pasture grass growth system. In particular, the invention isfurther directed to an herbicidal composition for use in modulating thegermination and growth of monocotyledonous and/or dicotyledenous and/orsedge weeds in a cereal growth system. In a particular embodiment, thecereal growth system is a non-rice cereal growth system comprising atleast one herbicide in which said herbicide is thaxtomin. Thecompositions of the present invention may further comprise a carrierand/or diluent. In a particular embodiment, the composition is anaqueous composition. In another particular embodiment, the thaxtomin inthe composition is dissolved in a diluent comprising an organic solventsuch as ethanol, isopropanol, or an aliphatic ketone such as acetone,methyl ethyl ketone, methyl isobutyl ketone.

In a related aspect, the invention is directed to the use of at leastone herbicidal agent, e.g., thaxtomin, in formulation of an herbicidefor modulating monocotyledonous and/or dicotyledenous and/or sedge weedsa cereal growth system, e.g., a non-rice cereal growth system.Similarly, the invention is directed to the use of at least oneherbicidal agent in formulation of an herbicide for modulatingmonocotyledonous and/or dicotyledenous and/or sedge weeds in a turfgrass growth system and/or Timothy grass growth system and/or pasturegrass growth system, wherein at least one herbicidal agent is thaxtomin.

The compositions of the present invention may comprise in addition tothaxtomin, at least one or more herbicides. Thus the invention maycomprise a thaxtomin and a chemical herbicide and/or bioherbicide.Compositions comprising thaxtomin and at least a second herbicide may beused in cereal growth systems (e.g., wheat, triticale, barley, oats,rye, corn, sorghum, sugarcane, rice or millet) and/or turf grass growthsystems and/or Timothy grass growth systems and/or pasture grass growthsystems.

Given that the invention is directed to the use of thaxtomin as a pre-or post-emergence herbicide, the invention is directed to a method forselectively modulating germination and growth of monocotyledonous,dicotyledonous and sedge weeds in a cereal crop growth system. In aparticular embodiment, the cereal growth system is a non-rice cerealcrop growth system comprising applying to said weeds or soil in saidcereal crop growing system at least one herbicidal agent, wherein saidherbicidal agent is thaxtomin, in an amount of effective to modulategermination and growth of said weeds but not modulate growth of cerealcrop in said cereal crop growth system. The cereal crop may include butis not limited to corn, wheat, triticale, barley, rye, oats, sorghum,sugarcane, and millet. The invention is further directed to a method formodulating germination and growth of monocotyledonous, dicotyledonousand sedge weeds in a turf, pasture and/or Timothy grass growth systemcomprising applying to said weeds or soil in said turf grass growingsystem at least one herbicidal agent, wherein said herbicidal agent isthaxtomin, in an amount of effective to modulate growth of said weedsbut not modulate germination and growth of turf grass in said turf grassgrowth system, pasture grass in said pasture grass growth system and/orTimothy grass in said Timothy grass growth system. The turf grass may beselected from the group consisting of Festuca sp., Poa sp., Bromus sp.,Lolium sp., Agrostis sp., Zoysia sp., Cynodon sp.

Further, the invention is directed to a method for modulatinggermination and growth of weeds selected from the group consisting ofChenopodium album, Abutilon theophrasti, Helianthus annuus, Ambrosiaartemesifolia, Amaranthus retroflexus, Convolvulus arvensis, Brassicakaber, Taraxacum officinale, Solanum nigrum, Malva neglect, Setarialutescens, Bromus tectorum, Poa annua, Poa pratensis, Lolium perenne L.var. Pace, Festuca arundinaceae Schreb. var. Aztec II, Anthem II,LS1100, Echinochloa crus-galli, comprising applying to said weeds orsoil an amount of thaxtomin or salt thereof effective to modulate saidgermination and growth of said weeds.

The method of the present invention may also involve the use of at leasta second herbicidal agent. The two herbicidal agents may be appliedtogether in one formulation or separately in two formulations. Controlof weeds can be achieved by using thaxtomin A in a tank mix or rotationwith other herbicidally active compounds known to have good activityagainst grass weeds but no or low phytotoxicity against cereal cropsand/or turf grass and/or, pasture grass and/or Timothy grasses. Inparticular, the invention relates to a method for modulating growth ofmonocotyledonous, dicotyledonous and sedge weeds comprising applying tosaid weeds an amount of thaxtomin and amount of at least a secondherbicidal agent to modulate growth of said weeds. The two herbicidalagents may be applied together in one formulation or separately in twoformulations. The thaxtomin and second herbicidal agent may be appliedin a cereal growth system (e.g., wheat, triticale, barley, oats, rye,corn, sorghum, sugarcane, rice or millet) and/or turf grass growthsystem and/or pasture grass growth system and/or Timothy grass growthsystem.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of Thaxtomin A.

DETAILED DESCRIPTION OF THE INVENTION

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Thaxtomin utilized in this invention may be derived in fermentation ofthe following actinomycetes cultures: S. scabies—ATCC 49173, S.acidiscabies—ATCC 49003 and BL37-EQ-010—or it can be purchased fromcommercial sources.

The thaxtomin utilized in the invention include but are not limited toagents described as cyclic dipeptides having the basic structurecyclo-(L-4-nitrotryptophyl-L-phenylalanyl). In embodiments, suitablediketopiperazine moieties may be N-methylated, and include congenerscarrying phenylalanyl alpha andring-carbon hydroxyl groups. The chemicalin a particular embodiment comprises:

-   -   wherein R₁ is methyl or H, R₂ is hydroxy or H, R₃ is methyl or        H, R₄ is hydroxy or H, R₅ is hydroxy or H, R₆ is hydroxy or H,        and combinations thereof.

Non limiting examples of suitable thaxtomin is for use in accordancewith the present invention include but are not limited to thaxtomin A,thaxtomin A ortho isomer, thaxtomin B, thaxtomin C, hydroxythaxtomin C,thaxtomin A p-isomer, hydroxythaxtomin A and des-N-methylthaxtomin C andderivatives of any of these (See FIG. 1).

The compositions of the present invention may be sprayed on the plant orapplied to soil. Particular embodiments are described in the Examples,infra. These compositions may be in the form of dust, coarse dust, microgranules, granules, wettable powder, emulsifiable concentrate, liquidpreparation, suspension concentrate, water degradable granules or oilsuspension.

The compositions of the invention do comprise a carrier and/or diluent.The term, ‘carrier’ as used herein means an inert, organic or inorganicmaterial, with which the active ingredient is mixed or formulated tofacilitate its application to plant or other object to be treated, orits storage, transport and/or handling. Examples of diluents or carriersfor the pre- and post-emergence herbicides include, but are not limitedto, water, milk, ethanol, mineral oil, glycerol.

The compositions of the present invention may comprise at least twoherbicidal agents. One herbicidal agent is thaxtomin set forth above. Itmay be present in one embodiment thaxtomin is present in an amountranging from about 0.01 to about 5.0 mg/mL. The other herbicidal agentmay be a bioherbicide and/or a chemical herbicide. The bioherbicide maybe selected from the group consisting of clove, cinnamon, lemongrass,citrus oils, orange peel oil, tentoxin, cornexistin, AAL-toxin,leptospermone, sarmentine, momilactone B, sorgoleone, ascaulatoxin andascaulatoxin aglycone. In a particular embodiment, the composition maycomprise thaxtomin, lemongrass oil and optionally a surfactant and/orvegetable oil. In another embodiment, the composition may comprisethaxtomin, sarmentine and optionally a nonionic surfactant and/orvegetable oil. The bioherbicide such as lemongrass oil or sarmentine maybe present in an amount ranging from about 0.1 mg/mL to about 50 mg/mLand more preferably between about 0.5 mg/mL to about 10 mg/mL Thechemical herbicide may be selected from the group consistingdiflufenzopyr and salts thereof, dicamba and salts thereof, topramezone,tembotrione, S-metolachlor, atrazine, mesotrione, primisulfuron-methyl,2,4-dichlorophenoxyacetic acid, nicosulfuron, thifensulfuron-methyl,asulam, metribuzin, diclofop-methyl, fluazifop, fenoxaprop-p-ethyl,asulam, oxyfluorfen, rimsulfuron, mecoprop, and quinclorac, thiobencarb,clomazone, cyhalofop, propanil, bensulfuron-methyl, penoxsulam,triclopyr, imazethapyr, halosulfuron-methyl, pendimethalin,bispyribac-sodium, carfentrazone ethyl, sodium bentazon/sodiumacifluorfen and orthosulfamuron.

The chemical herbicide such as pendimethalin or clomazone may be presentin a pre-emergent weed control application in an amount ranging fromabout 0.5 mg/mL to 15 mg/mL and a chemical herbicide such as cyhalofop,S-metolachlor, bispyribac-sodium, penoxsulam in a post-emergentapplication from about 1 mg/mL to about 40 mg/mL and more particularlybetween about 15 mg/ml to about 35 mg/mL. The composition may furthercomprise an adjuvant which may be vegetable oil comprising ethyl oleate,polyethylene dialkyl ester and ethoxylated nonylphenol. The compositionmay additionally comprise a surfactant to be used for the purpose ofemulsification, dispersion, wetting, spreading, integration,disintegration control, stabilization of active ingredients, improvementof fluidity or rust inhibition. The choice of dispersing and emulsifyingagents, such as non-ionic, anionic, amphoteric and cationic dispersingand emulsifying agents, and the amount employed is determined by thenature of the composition and the ability of the agent to facilitate thedispersion of the herbicidal compositions of the present invention.

For post-emergent formulations, the formulation components used maycontain smectite clays, attapulgite clays and similar swelling clays,thickeners such as xanthan gums, gum Arabic and other polysaccharidethickeners as well as dispersion stabilizers such as nonionicsurfactants (for example polyoxyethylene (20) monolaurate or polysorbate60 POE (20) sorbitan monostearate, ethylene glycol monostearate). Theconcentration of the clays may vary between about 0-2.5% w/w of thetotal formulation, the polysaccharide thickeners may range between about0-0.5% w/w of the total formulation and the surfactants may range fromabout 0-5% w/w of the total formulation.

Examples

The composition and method of the present invention will be furtherillustrated in the following, non-limiting Examples. The examples areillustrative of various embodiments only and do not limit the claimedinvention regarding the materials, conditions, weight ratios, processparameters and the like recited herein.

Example 1

In a pot study test in greenhouse conditions, 6-inch corn plants (Zeamays var. Sunglow) were sprayed with increasing concentrations ofthaxtomin A mixed in a carrier 4% ethanol, 0.02% polysorbate 60 POE (20)sorbitan monostearate solution. The spraying solutions contained 0.125,0.25, 0.5 and 1.0 mg thaxtomin A/mL, and the plants are sprayed untiltotal coverage. Each treatment was done in three replicates, and acontrol solution consists of water with 4% ethanol and 0.02% polysorbate60 POE (20) sorbitan monostearate as a surfactant. Prior to and aftertreatments, plants are grown in a greenhouse under artificial lights(12-h light/dark cycle) at 25° C.

Plants are evaluated in one-week intervals starting at 7 days aftertreatment. The final evaluation is done three weeks after treatment, atwhich time point, no phytotoxicity is observed in any of the test plantseven at the highest thaxtomin A concentration.

Example 2

A pot study is conducted to test the phytotoxicity of thaxtomin A oncorn (Zea mays var. Early Sunglow) and wheat (Triticum aestivum var.PR1404). To confirm the activity on broadleaf weeds, pigweed (Amaranthussp.) is planted in the same pot with either three corn or five wheatseeds, and sprayed simultaneously with the cereal test plants. The lessthan 3-inch tall plants grown under growth lights (12-h light/12-h dark)at 28° C. are sprayed with thaxtomin A solutions derived from a liquidculture of S. acidiscabies containing 0.5, and 1.0 mg thaxtomin A per mLof solvent (4% ethanol and 0.2% non-ionic surfactant). A solution of 4%ethanol+0.2% non-ionic surfactant without thaxtomin A is used as acontrol treatment. Each treatment is conducted in three replicates.Treated plants are kept at 28° C. under growth lights and observed atthree time points—7, 14 and 21 days after treatment—for visual symptomsof phytotoxicity on corn and wheat and % control of pigweed.

At each time point, no symptoms of phytotoxicity are observed in thecereal plants treated with thaxtomin A. The highest concentration ofthaxtomin A (1.0 mg/mL) results in a complete control of pigweed grownin the same pots with corn and wheat.

Example 3

To test the phytotoxicity of thaxtomin A on sorghum plants, five seedsof sorghum (Sorghum bicolor) are planted in each 4″×4″ plastic potfilled with soil. Plants were grown under optimal conditions in agreenhouse before and after treatment with solutions containing 0.5 and1.0 mg thaxtomin A/mL. At the time of the treatment, the plants areabout 3 inches tall. Each treatment is applied in three replicates, anda control treatment included plants treated with just the carrier (4%EtOH, 0.02% polysorbate 60 POE (20) sorbitan monostearate). Evaluationsfor phytotoxicity are performed at 7-day intervals starting one weekafter treatment. The last evaluation is performed three weeks after thetreatment at which point, no phytotoxicity is observed in the treatedplants in any treatment concentration.

Example 4

A strain of S. acidiscabies (ATCC-49003) is grown in oat bran broth for5 days (25° C., 200 rpm). The whole cell broth with thaxtomin A isextracted using XAD resin. The dried crude extract was resuspended in 4%ethanol and 0.02% non-ionic surfactant at a concentration of 10 mg/mL,and the solutions with two different concentrations of thaxtomin A (0.5and 1.0 mg/mL) are tested the following broadleaf weed species:

Lambsquarter—Chenopodium album

Velvetleaf—Abutilon theophrasti

Sunflower—Helianthus annuus

Ragweed, Common—Ambrosia artemesifolia

Pigweed, Redroot—Amaranthus retroflexus

Bindweed, Common—Convolvulus arvensis

Mustard, Wild—Brassica kaber

Dandelion—Taraxacum officinale

Nightshade, Black—Solanum nigrum

Mallow, Common—Malva neglecta

and on the following grass weed species:

Foxtail—Setaria lutescens

Brome, Downy—Bromus tectorum

Bluegrass, Annual—Poa annua

Bluegrass, Kentucky—Poa pratensis

Ryegrass, Perennial—(Lolium perenne L. var. Pace)

Fescue, Tall—(Festuca arundinaceae Schreb. var. Aztec II, Anthem II,LS1100)

Barnyard Grass—Echinochloa crus-galli

All plant species are tested in 4″×4″ plastic pots in three replicates.The untreated control plants are sprayed with the carrier solution (4%Ethanol, 0.02% glycosperse) and the positive control plants with Roundupat a rate corresponding to 1 fl. oz/acre. Treated plants are kept in agreenhouse under 12 h light/12 h dark conditions. Data for broadleafspecies from weekly evaluations are presented in Table 1.

TABLE 1 Weed control efficacy of a S. acidiscabies extract containingthaxtomin A on different weed species. Rating scale: 0 - no control, 1 -10% control, 2 - 25% control, 3 - 50% control, 4 - 75% control, 5 - 100%control. THAXTOMIN SOLUTION THAXTOMIN Weed UTC 0.5 mg/mL SOLUTION 1.0mg/mL species 7 DAYS 14 DAYS 21 DAYS 7 DAYS 14 DAYS 21 DAYS 7 DAYS 14DAYS 21 DAYS Dandelion 0.0 0.0 0.0 2.0 2.3 4.0 2.0 2.0 3.7 Nightshade0.0 0.0 0.0 2.7 2.2 2.3 2.7 2.0 2.3 Lambsquarter 0.0 0.0 0.0 2.0 2.0 2.02.0 2.0 2.0 Ragweed 0.0 0.0 0.0 1.0 0.5 0.0 1.0 0.5 0.0 Velvetleaf 0.00.0 0.0 1.7 1.0 1.0 2.0 1.0 0.3 Bindweed 0.0 0.0 0.0 1.0 1.0 0.0 1.2 1.00.0 Mustard 0.0 0.0 0.0 3.3 4.0 4.5 3.5 2.8 3.5 Sunflower 0.0 0.0 0.01.0 2.0 0.5 1.0 1.7 0.5 Mallow 0.0 0.0 0.0 1.0 1.0 1.0 1.2 1.0 1.0Pigweed 0.0 0.0 0.0 3.5 4.0 4.0 4.2 3.0 3.7

The extract from a bacterial culture of S. acidiscabies with a thaxtominA concentration of 0.5 mg/mL or higher showed good efficacy (>50%)against at least three of the most common broadleaf weed species(dandelion, mustard and pigweed) in both cereal and turf growingsystems. Control of some weeds such as Black nightshade and Commonlambsquarter was not complete but thaxtomin A even at the lowerconcentration (0.5 mg/mL) results in severe stunting of these weeds. Inthis same study, no adverse effects are observed in grass speciestreated with either 0.5 or 1.0 mg/mL thaxtomin A. In all tested grassspecies, no phytotoxic effects were visible at even the higher thaxtominA concentration.

Example 5

The combined effect of thaxtomin A and two commercial herbicides(Bipyribac-sodium formulated as Regiment and Lemongrass oil formulatedas GreenMatch EX) on small-flower umbrella sedge and watergrass istested in a field study using small (1-sq foot) plots. All singleproduct treatments and tank mix combinations were sprayed at 57 gal peracre. Evaluation of % control was done 14 days after treatment and theresults are presented in Table 2 below. Means in each column marked withthe same letter in Table 2 are not statistically different from eachother at p<0.05

According to the results, lemongrass oil at 1.25% weight does notimprove the efficacy of thaxtomin A (at 0.25 mg/mL) on sedge but itsignificantly increases the efficacy on grass weeds such as watergrass(field test) and sprangletop (greenhouse test).

TABLE 2 Effect of thaxtomin A alone and in combination withbispyribac-sodium and lemongrass oil on two rice weeds, small-flowerumbrella sedge and watergrass. Treatment Sedge control (%) Watergrasscontrol (%) Thaxtomin 0.25 mg/mL   95a   5d Thaxtomin 0.5 mg/mL  100a  5d Bispyribac-sodium 87.5a 32.5a (12 g/acre) Bispyribac-sodium ½ 47.5c  15c (6 g/acre) Bispyribac-sodium ½ + 67.5b   25ab Thaxtomin 0.5 mg/mLBispyribac-sodium ½ +   55bc  7.5c Thaxtomin 0.25 mg/mL Lemongrass oil5%   15d   10c Lemongrass oil 2.5% 12.5d   10c Lemongrass oil 1.25%  20d   5d Lemongrass oil 1.25% +  100a   10c Thax 0.25 mg/mL Lemongrassoil 1.25% +  100a   20b Thaxtomin 0.5 mg/mL

According to the results, lemongrass oil at 1.25% does not improve theefficacy of thaxtomin A (at 0.25 mg/mL) on sedge but it significantlyincreases the efficacy on grass weeds such as watergrass (field test)and sprangletop. Thaxtomin A (at 0.5 mg/mL) improves the efficacy of anALS inhibitor, bipyribac sodium; used at half label rate on both sedgeand grasses.

Example 6

The efficacy of thaxtomin A derived from a liquid culture of S.acidiscabies is tested in a field study on rice using 4.9 sq-ft plotssurrounded by a metal ring. Treatments with either thaxtomin A orthaxtomin A in combination with lemongrass oil (formulated as GreenMatchEX) or cyhalofop (formulated as Clincher CA) were done using a hand-heldsprayer with a water volume corresponding to 57 gallons per acre. Rice(variety M209) was grown until maturity and harvested by hand for yieldand weed count assessment. Results of yield (kg/ha), and numbers ofredstem, small-flower umbrella sedge, and sprangletop in each plot arepresented in Table 3 below.

TABLE 3 Effect of thaxtomin A alone and in combination with lemongrassoil and cyhalofop on rice yield and weed control. Treatment Yield(kg/ha) # of redstem # of sedge # of sprangletop 1  7516b 10.3 0.8a86.0a 2  7876b  0.5b 1.0a 76.0a 3  9054ab  0.3b 0.5a 69.3a 4 11296a12.8a 0.5a  4.0b 1. UTC; 2. Thaxtomin A (180 g/acre); 3. Lemongrass oil1.25% + thaxtomin A (90 g/acre); 4. Cyhalofop (half label rate; 52g/acre) + thaxtomin A (90 g/acre) + veg oil 2.5% Means in each columnmarked with the same letter are not statistically different from eachother at p < 0.05.

Results indicate that thaxtomin at 180 g/acre significantly reduced thenumber of sedges but had no effect on sprangletop or yield. When used athalf rate (thaxtomin A 90 g/acre), a combination with lemongrass oil hadbetter effect on sedges than a combination with cyhalofop (used at halflabel rate 52 g/acre). Good grass weed (sprangletop) control is achievedwhen thaxtomin (90 g/acre) is combined with cyhalofop at half the labelrate—this combination also improves the yield significantly.

Example 7

Cyhalofop (2-[4-(4-cyano-2-fluorophenoxy)phenoxy]propanoic acid, butylester) is also mixed together with adjuvant containing ethyl oleate,polyethylene dialky ester and ethoxylated nonylphenol (2.5% v/v) andincreasing concentrations of thaxtomin A (purified from the ATCC strain49003) at concentrations 0.1, 0.2 and 0.4 mg/ml. The concentrations ofthe 2-[4-(4-cyano-fluorophenoxy)phenoxy]propanoic acid, butyl esterbefore dilution are 29.6% (2.38 lb/gal) and 21.7% (2 lb/gal),respectively. The effect of these mixtures on the growth of common waterplantain, red stem, smallflower sedge and sprangletop is determined inthe greenhouse. Similarly, rice plants of variety M104 are grown andtested for phytotoxic effects, and all plants are evaluated 7, 14, and21 days after treatment. Results of from the study with cyhalofopformulated as Clincher CA at the 21-day evaluation point are presentedin Table 4 below.

TABLE 4 Effect of thaxtomin A alone and with cyhalofop on rice yield andweed control Clincher CA (6.5 oz/acre) + Thaxtomin A RedstemWaterplantain Sedge Sprangletop (mg/mL) % control % control % control %control UTC 0 0 0 0 0 - no thx A 75 8 0 90 0.1 100 85 87 100 0.2 97 8788 100 0.4 100 85 100 100As a conclusion, Clincher CA (29.6% cyhalofop by weight) applied at halflabel rate (6.5 oz/acre) has good efficacy against grass weeds—not sogood on broadleaves and poor on sedges. A combination of Clincher CA(cyhalofop) and thaxtomin A provides good control of all rice weedstested in this study. Efficacy of thaxtomin A against grass weeds issubstantially improved if combined with Clincher. Combination ofthaxtomin A with Clincher CA did not cause phytotoxicity on rice at anytested concentration.

Example 8

Penoxsulam(2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5c]pyrimidin-2-yl)-6-trifluoromethyl)benzenesulfonamide)is mixed together with adjuvant containing ethyl oleate, polyethylenedialky ester and ethoxylated nonylphenol (2.5% v/v) and increasingconcentrations of thaxtomin A (purified from the ATCC strain 49003) atconcentrations 0.1, 0.2 and 0.4 mg/ml. The concentrations of the2-[4-(4-cyano-fluorophenoxy)phenoxy]propanoic acid, butyl ester or2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5c]pyrimidin-2-yl)-6-trifluoromethyl)benzenesulfonamidebefore dilution are 29.6% (2.38 lb/gal) and 21.7% (2 lb/gal),respectively. The effect of these mixtures on the growth of common waterplantain, red stem, smallflower sedge and sprangletop is determined inthe greenhouse. Similarly, rice plants of variety M104 are grown andtested for phytotoxic effects, and all plants are evaluated 7, 14, and21 days after treatment.

Example 9

A strain of S. acidiscabies was grown in oat bran broth for 5 days (25°C., 200 rpm). The whole cell broth was extracted using XAD resin, andthe dried crude extract was resuspended in 4% ethanol and 0.2% non-ionicsurfactant at a concentration of 10 mg/mL. The diluted extractscontaining 0.2 and 0.4 mg thaxtomin A per mL were tested on three weedspecies (redstem; Ammania spp., smallflower umbrella sedge; Cyperusdifformis and sprangletop: Leptochloa uninervia). Other treatmentsincluded sarmentine at 2.5 and 5.0 mg/mL, and a combination treatmentcontaining 0.2 mg thaxtomin A and 2.5 mg sarmentine per mL. Eachtreatment was applied in three replicates. Treated plants were kept in agreenhouse under 12 h light/12 h dark conditions. Results from anevaluation performed 25 days after treatment are presented in Table 5.

TABLE 5 Efficacy of herbicidal treatments using thaxtomin A (0.2 and 0.4mg/mL) or sarmentine (2.5 or 5.0 mg/mL) alone or in combination (0.2 +2.5 mg/mL) to control broadleaf, sedge and grass weeds in a greenhousestudy. Control of Control of Control of treatment redstem (%) sedge (%)sprangletop (%) UTC   0a   0a   0a Thaxtomin A 0.2 mg/mL  5.0b 48.3b 8.3ab Thaxtomin A 0.4 mg/mL 11.7c* 91.7d 10.0b Thaxtomin 0.2 + 11.7c*61.7c 73.3c Sarmentine 2.5 mg/mL Sarmentine 2.5 mg/mL   0a  8.3a 80.0cSarmentine 5.0 mg/mL  2.5ab  6.7a 92.3d *stunted. In a column, Meansfollowed by the same letter are not statistically different from eachother at p < 0.05.

Thaxtomin A at the highest concentration of 0.4 mg/mL provides excellentcontrol of sedge but poor control of the grass weed (sprangletop). Whencombined with sarmentine, the efficacy against grass weeds improvessignificantly. Also, efficacy against sedge is improved with thecombination treatment compared with the single application of thaxtominA alone at the corresponding concentration. In this study, the controlof the broadleaf weed (redstem) is poor with all treatments.

Although this invention has been described with reference to specificembodiments, the details thereof are not to be construed as limiting, asit is obvious that one can use various equivalents, changes andmodifications and still be within the scope of the present invention.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

CITED REFERENCES

-   Beauséjour, J., C. Goyer, et al. (1999). “Production of thaxtomin A    by Streptomyces scabies strains in plant extract containing media.”    Can J Microbiol 45: 764-768.-   Duke, S. O., S. R. Baerson, et al. (2003). “United States Department    of Agriculture-Agricultural Research Service research on natural    products for pest management.” Pest Manag Sci 59: 708-717.-   Duke, S. O., F. E. Dayan, et al. (2000). “Natural products as    sources of herbicides: current status and future trends.” Weed    Research 40: 99-111.-   Fry, B. A. and R. Loria (2002). “Thaxtomin A: Evidence for a plant    cell wall target.” Physiological and Molecular Plant Pathology 60:    1-8.-   Gerwick, B. C., P. R. Graupner, et al. (2005). Methylidene    mevalonates and their use as herbicides. U. p. 7393812: 16.-   Healy, F. G., M. J. Wach, et al. (2000). “The txtAB genes of the    plant pathogen Streptomyces acidiscabies encode a peptide synthetase    required for phytotoxin thaxtomin A production and pathogenicity.”    Molecular Microbiology 38: 794-804.-   Hiltunen, L. H., I. Laakso, et al. (2006). “Influence of thaxtomins    in different combinations and concentrations on growth of    micropropagated potato shoot cultures.” J Agric Food Chem 54:    3372-3379.-   Hoagland, R. E. (2001). “Microbial allelochemicals and pathogens as    bioherbicidal agents.” Weed Technology 15: 835-857.-   Kang, Y., S. Semones, et al. (2008). Methods of controlling algae    with thaxtomin and thaxtomin compositions. USA, Novozymes    Biologicals, Inc.-   King, R. R., C. H. Lawrence, et al. (1992). “Chemistry of    phytotoxins associated with Streptomyces scabies, the causal    organism of potato common scab.” J. Agric. Food Chem 40: 834-837.-   King, R. R., C. H. Lawrence, et al. (1989). “Isolation and    characterization of phytotoxin associated with Streptomyces    scabies.” Journal of the Chemical Society, Chemical Communications    13: 849-850.-   King, R. R., C. H. Lawrence, et al. (2003). “More chemistry of the    thaxtomin phytotoxins.” Phytochemistry 64: 1091-1096.-   King, R. R., C. H. Lawrence, et al. (2001). “Herbicidal properties    of the thaxtomin group of phytotoxins.” J Agric Food Chem 49:    2298-2301.-   Loria, R., R. A. Bukhalid, et al. (1995). “Differential production    of thaxtomins by pathogenic Streptomyces species in vitro”    Phytopathology 85: 537-541.

1. A method for modulating germination and growth of monocotyledonous,dicotyledonous and sedge weeds in a non-rice cereal crop growth systemand/or a turf grass growth system and/or Timothy grass growth systemand/or pasture grass growth system, comprising applying to said weeds orsoil in said growth system at least one herbicidal agent, wherein saidherbicidal agent is thaxtomin, in an amount of effective to modulategrowth of said weeds but not modulate growth of non-rice cereal crop insaid non-rice cereal crop growth system and/or turf, pasture and/orTimothy grass in said turf, pasture and/or Timothy grass growth system.2. The method according to claim 1, wherein said cereal crop is at leastone of wheat, triticale, barley, oats, rye, corn, sorghum, sugarcane ormillet.
 3. The method according to claim 3, wherein said turf grass isselected from the group consisting of Festuca sp., Poa sp., Bromus sp.,Lolium sp., Agrostis sp., Zoysia sp., Cynodon sp.
 4. The methodaccording to claims 1, wherein the thaxtomin has the followingcomposition:

wherein R₁ is methyl or H, R₂ is hydroxy or H, R₃ is methyl or H, R₄ ishydroxy or H, R₅ is hydroxy or H, R₆ is hydroxy or H, and combinationsthereof.
 5. The method according to claims 1, wherein the weed isselected from the group consisting of Chenopodium album, Abutilontheophrasti, Helianthus annuus, Ambrosia artemesifolia, Amaranthusretroflexus, Convolvulus arvensis, Brassica kaber, Taraxacum officinale,Solanum nigrum, Malva neglect, Setaria lutescens, Bromus tectorum, Poaannua, Poa pratensis, Lolium perenne L. var. Pace, Festuca arundinaceaeSchreb. var. Aztec II, Anthem II, LS1100, Echinochloa crus-galli.
 6. Amethod for modulating growth of monocotyledonous, dicotyledonous andsedge weeds and aquatic weeds selected from the group consisting ofAmmania sp., Alisma plantago-aquatica, Cyperus sp., Leptochloa sp.comprising applying to said weeds or soil an amount of at least twoherbicidal agents effective to modulate growth of said weeds, wherein afirst herbicidal agent is thaxtomin.
 7. The method according to claim 6,wherein said monocotyledonous, dicotyledonous and sedge weeds aremodulated in a cereal growth system.
 8. The method according to claim 7,wherein the cereal growth system is a rice growth system.
 9. The methodaccording to claim 6, wherein said second herbicidal agent is a chemicalherbicide or bioherbicide.
 10. The method according to claim 6, whereinthe second herbicidal agent is a chemical herbicide selected from thegroup consisting of diflufenzopyr and salts thereof, dicamba and saltsthereof, topramezone, tembotrione, S-metolachlor, atrazine, mesotrione,primisulfuron-methyl, 2,4-dichlorophenoxyacetic acid, nicosulfuron,thifensulfuron-methyl, asulam, metribuzin, diclofop-methyl, fluazifop,fenoxaprop-p-ethyl, asulam, oxyfluorfen, rimsulfuron, mecoprop, andquinclorac, thiobencarb, clomazone, cyhalofop, propanil,bensulfuron-methyl, penoxsulam, triclopyr, imazethapyr,halosulfuron-methyl, pendimethalin, bispyribac-sodium, carfentrazoneethyl, sodium bentazon/sodium acifluorfen, orthosulfamuron.
 11. Themethod according to claim 6, wherein the second herbicidal agent is abioherbicide selected from the group consisting of clove, cinnamon,lemongrass, citrus oils, orange peel oil, tentoxin, cornexistin,AAL-toxin, leptospermone, sarmentine, momilactone B, sorgoleone,ascaulatoxin and ascaulatoxin aglycone.
 12. The method according toclaim 6, wherein the first and second herbicidal agents are applied inrotation.
 13. The method according to claim 6, wherein the first andsecond herbicidal agents are applied together.
 14. An herbicidalcomposition comprising at least two herbicides wherein a first herbicideis thaxtomin and a second herbicide is a bioherbicide or chemicalherbicide.
 15. The composition according to claims 14, wherein saidcomposition further comprises an adjuvant, a non-ionic surfactant and/oran organic solvent.
 16. The composition according to claims 14, whereinsaid composition further comprises a non-ionic surfactant and/or analiphatic alcohol.